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Team:BYU Provo/Notebook/Metabolism/febapr
From 2014.igem.org
BYU 2014 Notebook |
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Week of March 22nd
March 17, 2014
Researched articles on the effects of heavy metals on waste water treatment plants and the effects of heavy metals on bacteria to prepare for our presentation on N.multiformis metabolism optimization. Searched for common bacteria with heavy metal resistance genes that could be possibly insert into our bacterial chassis; finding sequences and reading about success rates in data of those that had been transferred.
March 20, 2014
Searched for the most commonly prescribed antibiotics in the United States. Top prescribed antibiotics include penicillins and macrolides according to the New England Journal of Medicine (2013) [http://www.nejm.org/doi/full/10.1056/NEJMc1212055#t=article]
March 21, 2014
Searched articles on the effectiveness of macrolide and beta-lactam degradation enzymes. Researched bacteria with a known gene sequences to degrade both types of antibiotics.
Week of March 29th, 2014
March 24, 2014
Investigated macrolide antibiotic degradation, settling on the ethryomycin esterase as the enzyme. Found part BBa_K1159000 in the IGEM registry which contains the Erythromycin Esterase Type II (EreB) gene that degrades macrolides.
March 28, 2014
Used the Anderson Promoter Collection to determine which promoters have the highest rate of expression. Antibiotic degradation genes would need medium to strong expression to be useful to the bacteria
Week of April 5th
April 1, 2014
Contacted the 2013 Technical University of Munich IGEM team to inquire about the EreB plasmid since because the registry said that it was not available. Received a response that the part would be available for 2014. Also contacted IGEM to request the part in the 2014 plate.
April 3, 2014
Researched scholarly articles about denitrifying genes to determine which particular enzymes are the most important. The paper describes several experiments with these enzymes in soil denitrifiers, the genes required to denitrify, and the importance of each gene present in soil bacteria. [http://link.springer.com/article/10.1007%2Fs00248-011-9909-5/fulltext.html]
April 4, 2014
Checked denitrifying genes for internal restriction enzyme sequences.
Week of April 12th, 2014
April 7, 2014
Prepared primer sequences to perform mutagenesis to exchange nucleotides and change the restriction site within the gene. Primers were designed for the denitrification norB gene that contained the IGEM plasmid restriction site EcoR1. Those primers were:
- 5’-CCGACCACGTACTGAAGGCCCATGATC-3’
- 5’-GATCATGGGCCTTCTGTACGTGGTCGG-3’
- 5’-TGCAGCCAGTCCTGTAGCACCCCG-3’
- 5’-CGGGGTGCTACAGGTCTGGCTGCA-3’
April 9, 2014
Finished the circuit write up for macrolide degradation and outlined a protocol to test the function of the gene. Following the write-up, we transformed the IGEM constitutive promoter BBaJ23109 to test its functionality in competent E.coli.
April 11, 2014
Performed plasmid preps with the transformed bacteria according to the common protocol.
